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Titolo:
Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture
Autore:
Berger, F; Soligo, D; Schwarz, K; Bossolasco, P; Schrezenmeier, H; Kubanek, B; Deliliers, GL; Licht, T;
Indirizzi:
Tech Univ Munich, Klinikum Rechts Isar, Med Klin 3, D-81675 Munich, Germany Tech Univ Munich Munich Germany D-81675 Klin 3, D-81675 Munich, Germany Osped Fatebenefratelli & Oftalm, Fdn Matarelli, Milan, Italy Osped Fatebenefratelli & Oftalm Milan Italy Fdn Matarelli, Milan, Italy Osped Maggiore, IRCCS, Bone Marrow Transplantat Unit, Milan, Italy Osped Maggiore Milan Italy Bone Marrow Transplantat Unit, Milan, Italy Free Univ Berlin, Klinikum Benjamin Franklin, D-12200 Berlin, Germany FreeUniv Berlin Berlin Germany D-12200 ranklin, D-12200 Berlin, Germany Univ Ulm Klinikum, Ulm, Germany Univ Ulm Klinikum Ulm GermanyUniv Ulm Klinikum, Ulm, Germany NCI, Mol Biol Lab, NIH, Bethesda, MD 20892 USA NCI Bethesda MD USA 20892NCI, Mol Biol Lab, NIH, Bethesda, MD 20892 USA
Titolo Testata:
GENE THERAPY
fascicolo: 9, volume: 8, anno: 2001,
pagine: 687 - 696
SICI:
0969-7128(200105)8:9<687:ERTOPH>2.0.ZU;2-R
Fonte:
ISI
Lingua:
ENG
Soggetto:
HEMATOPOIETIC STEM-CELLS; BONE-MARROW CELLS; COMBINED IMMUNODEFICIENT MICE; MULTIDRUG-RESISTANCE GENE; HUMAN MDR1 GENE; P-GLYCOPROTEIN EXPRESSION; GREEN FLUORESCENT PROTEIN; COLONY-STIMULATING FACTOR; LONG-TERM HEMATOPOIESIS; EX-VIVO EXPANSION;
Keywords:
hematopoietic stem cells; gene therapy; growth factors; rhodamine; multidrug resistance; P-glycoprotein;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
64
Recensione:
Indirizzi per estratti:
Indirizzo: Licht, T Tech Univ Munich, Klinikum Rechts Isar, Med Klin 3, Ismaninger Str 22, D-81675 Munich, Germany Tech Univ Munich Ismaninger Str 22 Munich Germany D-81675 ermany
Citazione:
F. Berger et al., "Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture", GENE THER, 8(9), 2001, pp. 687-696

Abstract

Retroviral transduction of hematopoietic cells has resulted in unsatisfactory gene marking in clinical studies. Since cytokine-stimulated stem cells have engrafted poorly in animal models, we investigated phenotypic changes during culture of peripheral blood progenitor cells (PBPC). Human CD34(+) HLA-DRlow cells, immunomagnetically separated from PBPC collections, were found to extrude rhodamine-123, which is characteristic for primitive hematopoietic cells. Cells were grown in suspension cultures supplemented with cytokines. While interleukin-3-containing factor combinations promoted cell proliferation they caused loss of rhodamine-123 extrusion and reduced the frequencies of cobblestone area-forming cells (CAFC), Several other cytokines failed to stimulate cell divisions, which are required for retroviral transduction. A combination including Flt-3 ligand (FL), interleukin-6 and stem cell factor (SCF) preserved an immature phenotype for 5 to 6 days and stimulated cell divisions, which was improved upon addition of leukemia inhibitory factor and interleukin-ll. Furthermore, the CAFC frequency among cells treated with these cytokines was increased as compared with widely used cocktails containing interleukin-3, interleukin-6 and SCF. Rhodamine-123 appeared to be a particularly sensitive indicator for differentiation of PBPC, For analysis of gene transfer, amphotropic retroviruses conferring an MDR1 cDNA were added repeatedly for 6 days to cytokine-treated PBPC stroma-free cultures. Proviral cDNA was detected by polymerase chain reaction in 68% of cobblestone areas derived from CD34(+) HLA-DRlow cells that had been exposedto Flt-3 ligand, interleukin-6 and SCF. In summary conditions were identified that facilitate efficient transduction of early PBPC with amphotropic retroviruses while preserving a primitive phenotype for extended periods.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 09/04/20 alle ore 19:55:01